GSM6934274: E_6Mo1_2853; Mus musculus; ncRNA-Seq
The mice were anesthetized by an intraperitoneal injection of ketamine (100 mg/kg bw; Ursotamin®; Serumwerke Bernburg, Bernburg, Germany) and xylazine (12 mg/kg bw; Rompun®; Bayer, Leverkusen, Germany). Subsequently, they were fixed on a heating pad in the supine position. After midline laparotomy, a maximal volume of blood (~700-1000 µL) was taken from the vena cava and transferred into plasma tubes (Sarstedt, Nümbrecht, Germany). The blood samples were then centrifuged at 20°C and 10.000 x g for 5 min to remove platelets, large vesicles, and cell debris; the resulting platelet-free plasma was stored at -80°C until further use. Two hundred microliters of mouse plasma was transferred to a 1 mL open-top thickwall polypropylene ultracentrifugation tube (Beckman-Coulter, USA) and diluted with 800 µL of phosphate-buffered saline to prevent the tube from collapsing in the ultracentrifuge vacuum. Samples were centrifuged for 2 h at 4°C at 100,000 x g using Type 50.4 Ti fixed-angle rotor (Beckmann-Coulter, USA). Supernatants were carefully removed, and the EV-containing pellets were resuspended in 20 µL of phosphate-buffered saline. Samples were stored at –80°C until further analyses. EV-enriched pellets further referred to as EV fractions and EV-depleted plasma referred to as the free circulating (fc) fraction were used for RNA isolation. All samples (blood from one animal corresponding to one sample) were treated separately, and no samples were pooled. EV- and fc- total RNAs were isolated semi-automated using the miRNeasy Micro kit (Qiagen, Hilden, Germany) and Qiacube isolation robot according to the manufacturer's recommendations with the addition of 2 µL RNase-free glycogen (20 mg/mL, Invitrogen, Carlsbad, CA, USA) to facilitate RNA precipitation. For each sample, at least two replicated sequencing results were available (12- and 18-month replicates, all other time points in triplicate). The RNA concentrations of the EV- and fc-fractions were measured using a Qubit™ microRNA Assay Kit. Isolated EV-RNA and fc-RNA samples were analyzed by Agilent small RNA chips, and 2 ng each (EV-RNA and fc RNA) was used for Illumina-compatible library preparation using the D-Plex Small RNA Kit (Diagenode, BE). The kits employ 3´-poly A tailing and template switch-based cDNA generation using unique molecular identifier (UMI)-tagged template switch oligos. After PCR amplification involving 13 cycles, libraries were purified from TBE-PAGEs. Illumina sequencing was carried out on a HiSeq2500 platform using the High Output mode for 96 cycles.